Compositions and methods to promote erythropoiesis

ABSTRACT

Described herein are compositions and methods for enhancing erythropoiesis in an individual in need thereof. Specifically agents that decrease the expression of Exosc8, Exosc9, Dis3, Dis3L or Exosc10, such as inhibitory nucleic acid molecules, produce an increase in red blood cell production in the individual.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/323,175filed on Jul. 3, 2014, which claims priority to U.S. ProvisionalApplication Ser. No. 61/842,569 filed on Jul. 3, 2014, which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under HL116365 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE DISCLOSURE

The present disclosure is related to compositions and methods to promoteerythropoiesis.

BACKGROUND

Maintenance of an adequate supply of oxygen to the body tissues isnecessary for survival. Because the oxygen-carrying capacity of blood isgoverned by the concentration of erythrocytes in the blood, theappropriate regulation of erythropoiesis, the process by which red bloodcells are produced, is critical.

Anemia is generally defined as a decrease in the number of red bloodcells or reduced hemoglobin in the blood, and can be caused by decreasederythropoiesis. Anemias associated with aging, infectious disease,chronic renal failure, end-stage renal disease, renal transplantation,cancer, AIDS, antiviral therapy, chronic stress, chemotherapy, radiationtherapy, bone marrow transplantation, and of genetic origin generallyrequire therapies to induce erythroid differentiation, and therefore toincrease red blood cell output. Currently, erythropoietin (Epo), aglycoprotein hormone that controls erythropoiesis, and itspharmacological derivatives are used to treat anemias such as anemiaresulting from chronic kidney disease. However, many anemias areEpo-insensitive. In addition, in certain contexts, Epo treatment hasbeen associated with undesirable side effects such as myocardialinfarction, stroke, venous thromboembolism and tumor recurrence. What isneeded are novel modes of stimulating erythropoiesis.

BRIEF SUMMARY

In one aspect, a method of enhancing erythropoiesis in an individual inneed thereof comprises administering an effective amount of an agentthat decreases the expression of Exosc8, Exosc9, DIS3, DIS3L or Exosc10,wherein the agent produces an increase in red blood cell production inthe individual.

In another aspect, a pharmaceutical composition comprises apharmaceutically acceptable excipient and a small interfering RNA thatreduces or inhibits the expression of Exosc8 (e.g., SEQ ID NO. 1 or 9),Exosc9 (e.g., SEQ ID NO. 2, 3 or 10), Dis 3 (e.g., SEQ ID Nos. 4, 5 or11), Dis3L (e.g., SEQ ID NOs. 6, 7, 12, 13, or 14), or Exosc10 (e.g.,SEQ ID NO. 8 or 15).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows representative TEM pictures of G1E-ER-GATA-1 cells with orwithout β-estradiol treatment. Scale bar, 2 μm.

FIG. 2 shows real-time RT-PCR analysis of mammalian ATG8 homologtranscript levels in bone marrow Ten 19+ cells from wild type andFoxo3^(−/−)mice (mean+/−SE; 6 independent biological samples).

FIG. 3 shows a ChIP analysis of Foxo3 and GATA-1 occupancy at selectautophagy genes and GATA-1 target gene loci in untreated and β-estradioltreated (24 hours) G1E-ER-GATA-1 cells (mean+/−SE; 4 independentexperiments).

FIG. 4 illustrates a GATA-1/Foxo3 network motif.

FIG. 5 shows Venn diagrams depicting the number of genes regulateduniquely or co-regulated by GATA-1 and Foxo3.

FIG. 6 shows Venn diagrams demonstrating relationships between GATA-1-and Foxo3-activated and -repressed genes.

FIG. 7 shows a gene ontology analysis of GATA-1/Foxo3-co-activatedgenes.

FIG. 8 shows a gene ontology analysis of GATA-1/Foxo3-co-repressedgenes.

FIG. 9 shows an RT-PCR analysis of Exosc8 expression upon GATA-1activation in G1E-ER-GATA-1 cells (mean+/−SE; 3 independentexperiments). Values were normalized to 18S rRNA expression.

FIG. 10 shows an RT-PCR analysis of Exosc8 mRNA level upon Foxo3knockdown in G1E-ER-GATA-1 cells (mean+/−SE; 4 independent experiments).Values were normalized to 18S rRNA expression.

FIG. 11 shows a ChIP-seq profile of GATA-1 occupancy at EXOSC8 inprimary human erythroblasts.

FIG. 12 illustrates the Exosc8 knockdown strategy in G1E-ER-GATA-1cells. IF, immunofluorescence.

FIG. 13 shows quantitative real-time RT-PCR analysis of mRNA levels incontrol versus Exosc8 knockdown G1E-ER-GATA-1 cells (mean+/−SE; 3independent experiments). Values were normalized to 18S rRNA expression.mRNA levels are shown relative to the control siRNA with estradioltreatment for activated genes (left) and without estradiol treatment forrepressed genes (middle). (Right) Fold changes upon Exosc8 knockdownrelative to control.

FIG. 14 shows quantitative real-time RT-PCR analysis of primarytranscripts in control versus Exosc8 knockdown G1E-ER-GATA-1 cells(mean+/−SE; 3 independent experiments). Values were normalized to 18SrRNA expression and the expression is shown relative to estradioltreated control siRNA. Fold changes are also depicted upon Exosc8knockdown relative to control (right). *, p<0.05; **, p<0.01; ***,p<0.001.

FIG. 15 shows a real-time RT-PCR analysis of Exosc8 and GATA-1-regulatedautophagy gene mRNA levels in control versus Exosc8 knockdownG1E-ER-GATA-1 cells (mean+/−SE; 3 independent experiments). Values werenormalized to 18S rRNA expression.

FIG. 16 shows semi-quantitative Western blot analysis of LC3B in Exosc8knockdown G1E-ER-GATA-1 cells. A representative image is shown from 3independent experiments.

FIG. 17 shows quantitation of cells containing LC3B-positiveautophagosomes (left) and the number of autophagosomes per cell (right)(mean+/−SE; 3 independent experiments).

FIG. 18 is a schematic diagram of the exosome complex.

FIG. 19 shows an expression profile of mRNA levels for exosome complexcomponents in primary mouse bone marrow erythroblasts during distinctmaturation stages mined from the Erythron DB. P, proerythroblast; B,basophilic erythroblast; O, orthochromatic erythroblast; R,reticulocyte.

FIG. 20 shows an expression profile of mRNAs encoding exosome complexcomponents during primary human erythroid differentiation mined from theHuman Erythroblast Maturation (HEM) Database. C, Colony-FormingUnit-Erythroid (CFU-E); P, proerythroblast; I, intermediate-stageerythroblast; L, late-stage erythroblast.

FIG. 21 shows quantitative real-time RT-PCR analysis of Exosc8 andselected GATA-1 target gene mRNA levels in control versus Exosc8knockdown primary murine erythroid precursor cells cultured underexpansion (−) or differentiation conditions (+) (mean+/−SE; 3independent experiments). Values were normalized to 18S rRNA expressionand the expression is shown relative to control shRNA under expansionconditions.

FIG. 22 shows flow cytometric quantitation of erythroid developmentalstage by CD71 and Ten 19 staining upon Exosc8 knockdown in primaryerythroid precursor cells. Representative flow cytometry data, with theR1-R5 gates denoted, is shown from 3 independent experiments. Thepercentage of live cells from each condition and the cell populations inR1-R5 stages are from 3 independent experiments (mean+/−SE). E,Expansion; D, Differentiation.

FIG. 23 shows representative images of Wright-Giemsa staining in controlversus Exosc8 shRNA infected primary erythroid precursor cells culturedin expansion or differentiation media (Scale bar, 10 μm) andquantitation of cell size by flow cytometry.

FIG. 24 shows representative images of LC3B punctae after Exosc8knockdown in primary erythroid precursor cells under expansion anddifferentiation culture conditions. LC3B-positive autophagosomes (left)containing cells where quantified and the number of autophagosomes percell (right) (mean+/−SE; 3 independent experiments).

FIG. 25 shows flow cytometric cell cycle analysis of primary erythroidprecursor cells infected with retrovirus expressing control or Exosc8 2shRNA. Representative cell cycle profile is shown from 3 independentexperiments. The percentage of the cell population in each cell cyclestage is from 3 independent experiments (mean+/−SE). Blue, S phase; Red,G₀/G₁ or G₂/M phase.

FIG. 26 shows quantitative ChIP analysis of serine 5-phosphorylated RNAPolymerase II occupancy at Exosc8-regulated GATA-1 target and controlgenes in control and Exosc8-knockdown primary murine erythroid precursorcells (mean+/−SE; 3 independent experiments). *, p<0.05; **, p<0.01;***, p<0.001.

FIG. 27 shows the crystal structure of the human exosome complexdemonstrating the interaction between Exosc8 and Exosc9.

FIG. 28 shows quantitative real-time RT-PCR analysis of Exosc9 andselected GATA-1 target gene mRNA levels in control versus Exosc9knockdown in expanding primary murine erythroid precursor cells(mean+/−SE; 4 independent experiments). Values were normalized to 18SrRNA level and the expression is shown relative to the control shRNAcondition.

FIG. 29 shows flow cytometric quantitation of erythroid maturation stageby CD71 and Ten 19 staining upon Exosc8 or Exosc9 knockdown in primaryerythroid precursor cells. Representative flow cytometry data, with theR1-R5 gates denoted, is shown from 4 independent experiments. Thepercentage of the cell populations in R1-R5 stages are an average of 4independent experiments.

FIG. 30 shows representative images of Wright-Giemsa stained cellsinfected with control versus Exosc8 or Exosc9 shRNA-expressing virus.Cells were cultured under expansion conditions (Scale bar, 10 μm). *,p<0.05; **, p<0.01; ***, p<0.001.

FIG. 31 shows flow cytometric quantitation of erythroid developmentalstage by CD71 and Ten 19 staining upon Exosc8 or Dis3 knockdown inprimary erythroid precursor cells. Representative flow cytometry data,with the R1-R5 gates denoted, is shown from 3 independent experiments.

FIG. 32 shows flow cytometric quantitation of erythroid developmentalstage by CD71 and Ten 19 staining upon Exosc8 or Dis3L knockdown inprimary erythroid precursor cells. Representative flow cytometry data,with the R1-R5 gates denoted, is shown from 3 independent experiments.

FIG. 33 shows the classification of GATA-1 and Exosc8 regulated genesbased on activation or repression.

FIG. 34 shows a gene ontology analysis of GATA-1-activated,Exosc8-repressed genes. Redundant Gene Ontology categories were curatedand removed.

FIG. 35 shows classification of Foxo3 and Exosc8 regulated genes basedon activation or repression.

FIG. 36 shows a gene ontology analysis of Foxo3-activated,Exosc8-repressed genes. Redundant Gene Ontology categories were curatedand removed.

FIG. 37 shows the name and function of genes in “cell cycle arrest”category derived from GO term analysis from GATA-1-activated andExosc8-repressed genes.

FIG. 38 shows quantitative real-time RT-PCR analysis of genes in “cellcycle arrest” category upon Exosc8 or Exosc9 knockdown in primary murineerythroid precursor cells under expansion conditions (mean+/−SE; 6independent experiments). Values were normalized to 18S rRNA and theexpression is shown relative to the control shRNA.

FIG. 39 shows ChIP-seq profiles of GATA-1 occupancy at cell cycleregulatory genes in primary human erythroblasts. All genes wereorientated left to right. *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 40 is a schematic showing that GATA-1/Foxo3 overcome the exosomecomplex-dependent erythroid maturation barricade.

FIG. 41 shows a real-time RT-PCR analysis of Exosc8 and exosome complexcatalytic subunits (Exosc10, Dis3, and Dis3l) mRNA levels in controlversus exosome complex component knockdown G1E-ER-GATA-1 cells(mean+/−SE; 3 independent experiments).

FIG. 42 shows representative GATA-1-regulated autophagy gene mRNA levelsin control versus exosome complex component knockdown G1E-ER-GATA-1cells (mean+/−SE; 3 independent experiments). Values were normalized to18S rRNA expression.

FIG. 43 shows semi-quantitative Western blot analysis of LC3B in exosomecomplex component knockdown G1E-ER-GATA-1 cells. A representative imageis shown from 3 independent experiments.

FIG. 44 shows real-time RT-PCR analysis of gene expression levels incontrol versus specific exosome complex component knockdownG1E-ER-GATA-1 cells (mean+/−SE; 3 independent experiments).

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Hematopoietic stem cells give rise to lineage-committed progenitors thatdifferentiate into erythroid precursors termed erythroblasts.Erythroblasts undergo sequential maturation steps, culminating inenucleation to yield reticulocytes and subsequently erythrocytes. Arestricted cohort of transcription factors, including GATA-1, KLF1, andSCL/TAL1 are responsible for establishing and maintaining the expressionof genes that endow the red cell with its unique identity. The presentinventors previously established that that the Forkhead transcriptionfactor Foxo3 amplifies GATA-1-mediated transcriptional activation ofgenes encoding components of the autophagy machinery. The Forkheadtranscription factor Foxo3 is essential for regulating oxidative stressresponse genes in erythroblasts. GATA-1 directly upregulates Foxo3expression, which in turn, amplifies GATA-1 activity to regulateautophagy gene expression.

Autophagy is the catabolic mechanism of cell degradation of cellularcomponents through the lysosomal machinery. Autophagy mediates cellularquality control and organelle remodeling, including the consumption ofmitochondria, as a critical determinant of physiological andpathological processes. A unique cellular structure, the autophagosome,engulfs damaged or aged organelles, such as mitochondria, and long-livedproteins. Following the regulated fusion of an autophagosome to alysosome, components within the autophagosome are degraded.

Autophagy is essential for multiple aspects of hematopoiesis, fromhematopoietic stem cell maintenance to the formation of specific bloodcell types. Nucleated erythroid precursor cells undergo sequentialmaturation steps culminating in erythroblast enucleation to yieldreticulocytes and subsequently erythrocytes. Autophagy-dependentdisposal of mitochondria (mitophagy) is a critical component of theerythroid maturation process.

Previously, the present inventors described a function of the masterregulator of erythropoiesis GATA-1 to transcriptionally upregulate genesencoding essential autophagy components. Dissecting how GATA-1instigates autophagy, the inventors discovered that GATA-1 and theforkhead transcription factor Foxo3 function combinatorially in afeed-forward loop to repress expression of a key component of theexosome complex. The exosome complex mediates degradation of aberrantpre-mRNAs and mRNAs and is required for the processing and degradationof pre-rRNAs, pre-snRNAs, and pre-tRNAs. In addition to mediating RNAdegradation, the exosome complex controls gene expression via regulatingtranscription start site usage, maintaining the heterochromatin markH3K9 methylation, and regulating expression of non-coding RNAs. A recentstudy revealed exosome complex and chromatin insulator componentco-occupancy at boundary elements and promoters in Drosophila. While theexosome complex is implicated in diverse molecular processes, whetherits functions contribute to or orchestrate specific biological processesis largely unexplored.

Specifically, through analyzing genetic networks regulatedcombinatorially by both GATA-1 and the forkhead protein FOX03, thepresent inventors discovered a feed-forward loop that promotesautophagy. GATA-1 induced FOX03 expression, and GATA-1 and FOX03co-occupied and regulated autophagy genes. Genomic analysis of theGATA-1/FOX03 target gene ensemble revealed GATA-1/FOX03-dependentrepression of Exosc8 (Rrp43), which encodes a pivotal component of theexosome that mediates RNA surveillance and epigenetic regulation.Strikingly, using shRNA to reduce Exosc8 or Exosc9 (Rrp45) expression inprimary erythroid precursor cells induced autophagy and erythroid cellmaturation. The exosome consists of nine essential core subunits and twocatalytic subunits. The inventors knocked down Exosc8, Exosc9 andcatalytic subunits, Dis3 (Rrp44), Dis3L and Exosc10 (Rrp6). Knockingdown Dis3, Dis3L and Exosc10 had qualitatively similar butquantitatively reduced results as knocking down Exosc8 on red blood cellmaturation.

More specifically, loss-of-function analysis revealed a mechanism inwhich the exosome components Exosc8 and Exosc9 create a blockade toprimary erythroid cell maturation. Disruption of this blockade inducedspontaneous erythroid maturation to yield late-stage erythroblasts andreticulocytes. The exosome complex had not been linked to hematopoiesisor erythroid cell biology, and therefore the results establish importantbiological functions for the exosome complex.

In one aspect, the inventors of the present application have discoveredthat reducing the levels of cellular RNA processing components of theRNA processing nuclear exosome (EXOSC8 and EXOSC9) stimulate theerythroid differentiation of primary erythroid cells. This finding hasalso been demonstrated with an erythroid cell line that mimics thenormal physiology of primary erythroid cells. This stimulation ofdifferentiation does not involve altered levels of the master regulatorof erythropoiesis, GATA-1. The enhanced maturation was demonstratedusing 1) RNA expression that provides a read-out for maturation and 2)Flow cytometric evaluation of erythroid surface protein expression thatprovides a read-out for maturation. Enhanced maturation was alsodemonstrated using morphology and cell cycle analysis. This molecularand cell biological evidence provides extremely strong evidence thatlowering the levels of EXOSC8, EXOSC9, DIS3, DIS3L and EXOSC10 stronglyinduces erythroid maturation, promoting erythropoiesis.

In one embodiment, a method of enhancing erythropoiesis in an individualin need thereof comprises administering an effective amount of an agentthat decreases the expression and/or activity of Exosc8, Exosc9, Dis3,Dis3L or Exosc10, wherein the agent produces an increase in red bloodcell production in the individual.

As used herein “decreases” means a reduction of at least 5% relative toa reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%,or even by as much as 75%, 85%, 95% or more.

As used herein “increases” means a stimulation of at least 5% relativeto a reference level. An increase may be by 5%, 10%, 15%, 20%, 25% or50%, or even by as much as 75%, 85%, 95% or more.

An “effective amount” is the amount of an agent required to amelioratethe symptoms of a disease or slow, stabilize, prevent, or reduce theseverity of the pathology in a subject relative to an untreated subject.The effective amount of active agent(s) varies depending upon the mannerof administration, the age, body weight, and general health of thesubject. Ultimately, the attending physician or veterinarian will decidethe appropriate amount and dosage regimen. Such amount is referred to asan “effective” amount.

The sequences of Exosc8, Exosc9, Dis3, Dis3L and Exosc10 are known inthe art. Human Exosc8 has accession number NM_181503.2 (SEQ ID NO. 1);human Exosc9 has 2 transcript variants, Exosc9 variant 1 has accessionnumber NM_001034194.1 (SEQ ID NO. 2), Exosc9 variant 2 has accessionnumber NM_005033.2 (SEQ ID NO. 3); human Dis3 has 2 transcript variants,Dis3 variant 1 has accession number NM_014953.4 (SEQ ID No. 4), Dis3variant 2 has accession number NM_001128226.2 (SEQ ID No. 5); humanDis3L has 2 transcript variants, Dis3L variant 1 has accession numberNM_001143688.1 (SEQ ID No. 6), Dis3L variant 2 has accession numberNM_133375.3 (SEQ ID No. 7); and human Exosc10 has accession numberNM_001001998.1 (SEQ ID NO. 8). Mouse Exosc8 has accession numberNM_001163570.1 (SEQ ID NO. 9); mouse Exosc9 has accession numberNM_019393.2 (SEQ ID NO. 10); mouse Dis3 has accession number NM_028315.2(SEQ ID No. 11); mouse Dis3L has 3 transcript variants, Dis3L variant 1has accession number NM_001001295 (SEQ ID No. 12), Dis3L variant 2 hasaccession number NM_172519.3 (SEQ ID No. 13), Dis3L variant 3 hasaccession number NM_001177784. 1 (SEQ ID No. 14); and mouse Exosc10 hasaccession number NM_016699.2 (SEQ ID NO. 15).

The term individual includes humans and other mammals, specificallyhumans.

In one aspect, the individual is a human in need of treatment for ananemic disorder, hemophilia, thalassemia, sickle cell disease, bonemarrow transplantation, hematopoietic stem cell transplantation,thrombocytopenia, pancytopenia, or hypoxia.

As used herein, anemia is a decrease in the number of red blood cellscaused by decreased production or increased destruction of red bloodcells. Anemia can be characterized by a reduced hematocrit level, thevolume % of red blood cells in the blood, also referred to as the packedcell volume (PCV) or the erythrocyte volume fraction (EVF). Thehematocrit varies with age and gender. For children, anemia is generallydefined as a hematocrit below about 35%, for adult non-pregnant womenbelow about 36%, for adult pregnant women below about 33%, and for adultmen below about 39%. In one aspect, the individual with an anemicdisorder has a reduced hematocrit.

Anemic disorders are associated with aging, infectious disease, chronicrenal failure, end-stage renal disease, renal transplantation, cancer,AIDS (e.g., zidovudine-induced anemia), antiviral therapy, chronicstress, chemotherapy, radiation therapy, bone marrow transplantation,nutritional iron deficiency, blood-loss such as preparation for surgerywith a high risk of blood loss, hemolysis, and are of genetic originsuch as congenital dyserythropoietic anemia.

Exemplary agents that decrease the expression and/or activity of Exosc8,Exosc9, Dis3, Dis3L or Exosc10 include inhibitory nucleic acids.

In one aspect, the agent that decreases the expression of Exosc8,Exosc9, Dis3, Dis3L or Exosc10 is an inhibitory nucleic acid molecule,wherein administration of the inhibitory nucleic acid moleculeselectively decreases the expression of Exosc8, Exosc9, Dis3, Dis3L orExosc10. The term “inhibitory nucleic acid molecule” means a singlestranded or double-stranded RNA or DNA, specifically RNA, such astriplex oligonucleotides, ribozymes, aptamers, small interfering RNAincluding siRNA (short interfering RNA) and shRNA (short hairpin RNA),antisense RNA, or a portion thereof, or an analog or mimetic thereof,that is capable of reducing or inhibiting the expression of a targetgene or sequence. Inhibitory nucleic acids can act by, for example,mediating the degradation or inhibiting the translation of mRNAs whichare complementary to the interfering RNA sequence. An inhibitory nucleicacid, when administered to a mammalian cell, results in a decrease(e.g., by 5%, 10%, 25%, 50%, 75%, or even 90-100%) in the expression(e.g., transcription or translation) of a target sequence. Typically, anucleic acid inhibitor comprises or corresponds to at least a portion ofa target nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule. Inhibitory nucleic acids may have substantial or completeidentity to the target gene or sequence, or may include a region ofmismatch (i.e., a mismatch motif). The sequence of the inhibitorynucleic acid can correspond to the full-length target gene, or asubsequence thereof. In one aspect, the inhibitory nucleic acidmolecules are chemically synthesized.

The specific sequence utilized in design of the inhibitory nucleic acidsis a contiguous sequence of nucleotides contained within the expressedgene message of the target. Factors that govern a target site for theinhibitory nucleic acid sequence include the length of the nucleic acid,binding affinity, and accessibility of the target sequence. Sequencesmay be screened in vitro for potency of their inhibitory activity bymeasuring inhibition of target protein translation and target relatedphenotype, e.g., inhibition of cell proliferation in cells in culture.In general it is known that most regions of the RNA (5′ and 3′untranslated regions, AUG initiation, coding, splice junctions andintrons) can be targeted using antisense oligonucleotides. Programs andalgorithms, known in the art, may be used to select appropriate targetsequences. In addition, optimal sequences may be selected utilizingprograms designed to predict the secondary structure of a specifiedsingle stranded nucleic acid sequence and allowing selection of thosesequences likely to occur in exposed single stranded regions of a foldedmRNA. Methods and compositions for designing appropriateoligonucleotides may be found, for example, in U.S. Pat. No. 6,251,588,the contents of which are incorporated herein by reference.

Phosphorothioate antisense oligonucleotides may be used. Modificationsof the phosphodiester linkage as well as of the heterocycle or the sugarmay provide an increase in efficiency. Phosphorothioate is used tomodify the phosphodiester linkage. An N3′-P5′ phosphoramidate linkagehas been described as stabilizing oligonucleotides to nucleases andincreasing the binding to RNA. A peptide nucleic acid (PNA) linkage is acomplete replacement of the ribose and phosphodiester backbone and isstable to nucleases, increases the binding affinity to RNA, and does notallow cleavage by RNAse H. Its basic structure is also amenable tomodifications that may allow its optimization as an antisense component.With respect to modifications of the heterocycle, certain heterocyclemodifications have proven to augment antisense effects withoutinterfering with RNAse H activity. An example of such modification isC-5 thiazole modification. Finally, modification of the sugar may alsobe considered. 2′-O-propyl and 2′-methoxyethoxy ribose modificationsstabilize oligonucleotides to nucleases in cell culture and in vivo.

Short interfering (si) RNA technology (also known as RNAi) generallyinvolves degradation of an mRNA of a particular sequence induced bydouble-stranded RNA (dsRNA) that is homologous to that sequence, thereby“interfering” with expression of the corresponding gene. A selected genemay be repressed by introducing a dsRNA which corresponds to all or asubstantial part of the mRNA for that gene. Without being held totheory, it is believed that when a long dsRNA is expressed, it isinitially processed by a ribonuclease III into shorter dsRNAoligonucleotides of as few as 21 to 22 base pairs in length.Accordingly, siRNA may be effected by introduction or expression ofrelatively short homologous dsRNAs. Exemplary siRNAs have sense andantisense strands of about 21 nucleotides that form approximately 19nucleotides of double stranded RNA with overhangs of two nucleotides ateach 3′ end.

siRNA has proven to be an effective means of decreasing gene expressionin a variety of cell types. siRNA typically decreases expression of agene to lower levels than that achieved using antisense techniques, andfrequently eliminates expression entirely. In mammalian cells, siRNAsare effective at concentrations that are several orders of magnitudebelow the concentrations typically used in antisense experiments.

The double stranded oligonucleotides used to effect RNAi arespecifically less than 30 base pairs in length, for example, about 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, or 17 base pairs or less inlength, and contain a segment sufficiently complementary to the targetmRNA to allow hybridization to the target mRNA. Optionally, the dsRNAoligonucleotide includes 3′ overhang ends. Exemplary 2-nucleotide 3′overhangs are composed of ribonucleotide residues of any type and may becomposed of 2′-deoxythymidine residues, which lowers the cost of RNAsynthesis and may enhance nuclease resistance of siRNAs in the cellculture medium and within transfected cells. Exemplary dsRNAs aresynthesized chemically or produced in vitro or in vivo using appropriateexpression vectors. Longer RNAs may be transcribed from promoters, suchas T7 RNA polymerase promoters, known in the art.

Longer dsRNAs of 50, 75, 100, or even 500 base pairs or more also may beutilized in certain embodiments. Exemplary concentrations of dsRNAs foreffecting RNAi are about 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 1.5 nM, 25 nM,or 100 nM, although other concentrations may be utilized depending uponthe nature of the cells treated, the gene target and other factorsreadily identifies by one of ordinary skill in the art.

Compared to siRNA, shRNA offers advantages in silencing longevity anddelivery options. Vectors that produce shRNAs, which are processedintracellularly into short duplex RNAs having siRNA-like propertiesprovide a renewable source of a gene-silencing reagent that can mediatepersistent gene silencing after stable integration of the vector intothe host-cell genome. Furthermore, the core silencing ‘hairpin’ cassettecan be readily inserted into retroviral, lentiviral, or adenoviralvectors, facilitating delivery of shRNAs into a broad range of celltypes.

A hairpin can be organized in either a left-handed hairpin (i.e.,5′-antisense-loop-sense-3′) or a right-handed hairpin (i.e.,5′-sense-loop-antisense-3′). The shRNA may also contain overhangs ateither the 5′ or 3′ end of either the sense strand or the antisensestrand, depending upon the organization of the hairpin. If there are anyoverhangs, they are specifically on the 3′ end of the hairpin andinclude 1 to 6 bases. The overhangs can be unmodified, or can containone or more specificity or stabilizing modifications, such as a halogenor O-alkyl modification of the 2′ position, or internucleotidemodifications such as phosphorothioate, phosphorodithioate, ormethylphosphonate modifications. The overhangs can be ribonucleic acid,deoxyribonucleic acid, or a combination of ribonucleic acid anddeoxyribonucleic acid.

Additionally, a hairpin can further comprise a phosphate group on the5′-most nucleotide. The phosphorylation of the 5′-most nucleotide refersto the presence of one or more phosphate groups attached to the 5′carbon of the sugar moiety of the 5′-terminal nucleotide. Specifically,there is only one phosphate group on the 5′ end of the region that willform the antisense strand following Dicer processing. In one exemplaryembodiment, a right-handed hairpin can include a 5′ end (i.e., the free5′ end of the sense region) that does not have a 5′ phosphate group, orcan have the 5′ carbon of the free 5′-most nucleotide of the senseregion being modified in such a way that prevents phosphorylation. Thiscan be achieved by a variety of methods including, but not limited to,addition of a phosphorylation blocking group (e.g., a 5′-O-alkyl group),or elimination of the 5′-OH functional group (e.g., the 5′-mostnucleotide is a 5′-deoxy nucleotide). In cases where the hairpin is aleft-handed hairpin, preferably the 5′ carbon position of the 5′-mostnucleotide is phosphorylated.

Hairpins that have stem lengths longer than 26 base pairs can beprocessed by Dicer such that some portions are not part of the resultingsiRNA that facilitates mRNA degradation. Accordingly the first region,which may include sense nucleotides, and the second region, which mayinclude antisense nucleotides, may also contain a stretch of nucleotidesthat are complementary (or at least substantially complementary to eachother), but are or are not the same as or complementary to the targetmRNA. While the stem of the shRNA can include complementary or partiallycomplementary antisense and sense strands exclusive of overhangs, theshRNA can also include the following: (1) the portion of the moleculethat is distal to the eventual Dicer cut site contains a region that issubstantially complementary/homologous to the target mRNA; and (2) theregion of the stem that is proximal to the Dicer cut site (i.e., theregion adjacent to the loop) is unrelated or only partially related(e.g., complementary/homologous) to the target mRNA. The nucleotidecontent of this second region can be chosen based on a number ofparameters including but not limited to thermodynamic traits orprofiles.

Modified shRNAs can retain the modifications in the post-Dicer processedduplex. In exemplary embodiments, in cases in which the hairpin is aright handed hairpin (e.g., 5′-S-loop-AS-3′) containing 2-6 nucleotideoverhangs on the 3′ end of the molecule, 2′-O-methyl modifications canbe added to nucleotides at position 2, positions 1 and 2, or positions1, 2, and 3 at the 5′ end of the hairpin. Also, Dicer processing ofhairpins with this configuration can retain the 5′ end of the sensestrand intact, thus preserving the pattern of chemical modification inthe post-Dicer processed duplex. Presence of a 3′ overhang in thisconfiguration can be particularly advantageous since blunt endedmolecules containing the prescribed modification pattern can be furtherprocessed by Dicer in such a way that the nucleotides carrying the 2′modifications are removed. In cases where the 3′ overhang ispresent/retained, the resulting duplex carrying the sense-modifiednucleotides can have highly favorable traits with respect to silencingspecificity and functionality. Examples of exemplary modificationpatterns are described in detail in U.S. Patent Publication No.20050223427 and International Patent Publication Nos. WO 2004/090105 andWO 2005/078094, the disclosures of each of which are incorporated byreference herein in their entirety.

shRNA may comprise sequences that were selected at random, or accordingto a rational design selection procedure. For example, rational designalgorithms are described in International Patent Publication No. WO2004/045543 and U.S. Patent Publication No. 20050255487, the disclosuresof which are incorporated herein by reference in their entireties.Additionally, it may be desirable to select sequences in whole or inpart based on average internal stability profiles (“AISPs”) or regionalinternal stability profiles (“RISPs”) that may facilitate access orprocessing by cellular machinery.

Ribozymes are enzymatic RNA molecules capable of catalyzing specificcleavage of mRNA, thus preventing translation. The mechanism of ribozymeaction involves sequence specific hybridization of the ribozyme moleculeto complementary target RNA, followed by an endonucleolytic cleavageevent. The ribozyme molecules specifically include (1) one or moresequences complementary to a target mRNA, and (2) the well-knowncatalytic sequence responsible for mRNA cleavage or a functionallyequivalent sequence (see, e.g., U.S. Pat. No. 5,093,246, which isincorporated herein by reference in its entirety).

While ribozymes that cleave mRNA at site-specific recognition sequencescan be used to destroy target mRNAs, hammerhead ribozymes mayalternatively be used. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. Specifically, the target mRNA has the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully in U.S.Pat. No. 5,633,133, the contents of which are incorporated herein byreference.

Gene targeting ribozymes may contain a hybridizing region complementaryto two regions of a target mRNA, each of which is at least 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides(but which need not both be the same length).

Hammerhead ribozyme sequences can be embedded in a stable RNA such as atransfer RNA (tRNA) to increase cleavage efficiency in vivo. Inparticular, RNA polymerase III-mediated expression of tRNA fusionribozymes is well known in the art. There are typically a number ofpotential hammerhead ribozyme cleavage sites within a given target cDNAsequence. Specifically, the ribozyme is engineered so that the cleavagerecognition site is located near the 5′ end of the target mRNA toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts. Furthermore, the use of any cleavagerecognition site located in the target sequence encoding differentportions of the target mRNA would allow the selective targeting of oneor the other target genes.

Ribozymes also include RNA endoribonucleases (“Cech-type ribozymes”)such as the one which occurs naturally in Tetrahymena thermophile,described in International Patent Publication No. WO 88/04300. TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence where after cleavage of the target RNA takesplace. In one embodiment, Cech-type ribozymes target eight base-pairactive site sequences that are present in a target gene or nucleic acidsequence.

Ribozymes can be composed of modified oligonucleotides (e.g., forimproved stability, targeting, etc.) and can be chemically synthesizedor produced through an expression vector. Because ribozymes, unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency. Additionally, in certain embodiments, aribozyme may be designed by first identifying a sequence portionsufficient to cause effective knockdown by RNAi. Portions of the samesequence may then be incorporated into a ribozyme.

Alternatively, target gene expression can be reduced by targetingdeoxyribonucleotide sequences complementary to the regulatory region ofthe gene (i.e., the promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the gene in target cells in thebody. Nucleic acid molecules to be used in triple helix formation forthe inhibition of transcription are specifically single stranded andcomposed of deoxyribonucleotides. The base composition of theseoligonucleotides should promote triple helix formation via Hoogsteenbase pairing rules, which generally require sizable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the target sequences that can be targeted for triplehelix formation may be increased by creating a so-called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Inhibitory nucleic acids can be administered directly or delivered tocells by transformation or transfection via a vector, including viralvectors or plasmids, into which has been placed DNA encoding theinhibitory oligonucleotide with the appropriate regulatory sequences,including a promoter, to result in expression of the inhibitoryoligonucleotide in the desired cell. Known methods include standardtransient transfection, stable transfection and delivery using virusesranging from retroviruses to adenoviruses. Delivery of nucleic acidinhibitors by replicating or replication-deficient vectors iscontemplated. Expression can also be driven by either constitutive orinducible promoter systems. In other embodiments, expression may beunder the control of tissue or development-specific promoters.

Vectors may be introduced by transfection using carrier compositionssuch as Lipofectamine 2000 (Life Technologies) or Oligofectamine™ (LifeTechnologies). Transfection efficiency may be checked using fluorescencemicroscopy for mammalian cell lines after co-transfection ofhGFP-encoding pAD3.

The effectiveness of the inhibitory oligonucleotide may be assessed byany of a number of assays, including reverse transcriptase polymerasechain reaction or Northern blot analysis to determine the level ofexisting human sclerostin mRNA, or Western blot analysis usingantibodies which recognize the human sclerostin protein, aftersufficient time for turnover of the endogenous pool after new proteinsynthesis is repressed.

In one embodiment, the inhibitory nucleic acid is an siRNA or an shRNAcomprising 19 to 29 nucleotides that are substantially complementary toa sequence of 19 to 29 nucleotides of Exosc8 (SEQ ID NO. 1 or 9),specifically in the region of nucleotides 363 to 405, nucleotides1129-1150, nucleotides 712-730, nucleotides 451 to 469 or nucleotides501 to 519 of SEQ ID NO. 9 and the corresponding regions of SEQ IDNO. 1. Within the region of nucleotides 363 to 405, the regions ofnucleotides 363 to 381, 384 to 405 or 371 to 392 may be employed.

Exemplary siRNAs for Exosc8 (SEQ ID NO. 9) include GAAAGAGGAUUUAUGCAUU(nucleotides 712-730, SEQ ID NO. 16), CAACAUAGGUUCAAUCAGU (nucleotides451-469, SEQ ID NO. 17), UGGAGCCGCUGGAGUAUUA (nucleotides 363-381, SEQID NO. 18), GGAAUACCACGGUCAUUUG (nucleotides 501-519, SEQ ID NO. 19),the corresponding nucleotides of SEQ ID NO. 1, and combinations thereof.

In one aspect, shRNAs that inhibit Exosc8 (SEQ ID NO. 9) include:

(nucleotides 384-405, SEQ ID NO. 20)TGCTGTTGACAGTGAGCGCCAGGAGATTTCTGAAGGAAAATAGTGAAGCCACAGATGTATTTTCCTTCAGAAATCTCCTGTTGCCTACTGCCTCGGA(nucleotides 371-392, SEQ ID NO. 21)TGCTGTTGACAGTGAGCGACGCTGGAGTATTACAGGAGATTAGTGAAGCCACAGATGTAATCTCCTGTAATACTCCAGCGGTGCCTACTGCCTCGGA(nucleotides 1129-1150, SEQ ID NO. 22)TGCTGTTGACAGTGAGCGCCACAAAGAAGTGAGCAAGCTATAGTGAAGCCACAGATGTATAGCTTGCTCACTTCTTTGTGTTGCCTACTGCCTCGGAin SEQ ID NO. 9, and the corresponding regions of SEQ ID NO. 1.

In one embodiment, the inhibitory nucleic acid is a siRNA or a shRNAcomprising 19 to 29 nucleotides that are substantially complementary toa sequence of 19 to 29 nucleotides of Exosc9 (SEQ ID NO. 2, 3 or 10). Inone aspect, shRNAs that inhibit Exosc9 include:

(nucleotides 1094-1116 SEQ ID NO. 23)TGCTGTTGACAGTGAGCGACAGATTGGAGACGGAATAGAATAGTGAAGCCACAGATGTATTCTATTCCGTCTCCAATCTGGTGCCTACTGCCTCGGA(nucleotides 1379-1401 SEQ ID NO. 24)TGCTGTTGACAGTGAGCGAAAGAAGAGAACTGCTAACTAATAGTGAAGCCACAGATGTATTAGTTAGCAGTTCTCTTCTTCTGCCTACTGCCTCGGAin SEQ ID NO. 10, and the corresponding regions of SEQ ID NO. 2 or 3.

In one embodiment, the inhibitory nucleic acid is a siRNA or a shRNAcomprising 19 to 29 nucleotides that are substantially complementary toa sequence of 19 to 29 nucleotides of Dis3 (SEQ ID NO. 4, 5 or 11). Inone aspect, shRNAs that inhibit Dis3 include

(nucleotides 2887-2909 SEQ ID NO. 25)TGCTGTTGACAGTGAGCGCCCACAGATCCCAGGAATAAATTAGTGAAGCCACAGATGTAATTTATTCCTGGGATCTGTGGTTGCCTACTGCCTCGGA(nucleotides 593-615 SEQ ID NO. 26)TGCTGTTGACAGTGAGCGACAGACAGTCAGCTGCAAGTTATAGTGAAGCCACAGATGTATAACTTGCAGCTGACTGTCTGCTGCCTACTGCCTCGGAin SEQ ID NO 11 or the corresponding regions of SEQ ID NOs. 4 or 5.

In one embodiment, the inhibitory nucleic acid is an siRNA or an shRNAcomprising 19 to 29 nucleotides that are substantially complementary toa sequence of 19 to 29 nucleotides of Dis3L (SEQ ID NO. 6, 7, 12, 13 or14). In one aspect, shRNAs that inhibit Dis3L include:

(nucleotides 414-436 SEQ ID NO. 27)TGCTGTTGACAGTGAGCGCGGCCGGAGACAGTATAACAAATAGTGAAGCCACAGATGTATTTGTTATACTGTCTCCGGCCTTGCCTACTGCCTCGGA(nucleotides 1677-1699 SEQ ID NO. 28)TGCTGTTGACAGTGAGCGCTACATCGATGTTGAAGCTAGATAGTGAAGCCACAGATGTATCTAGCTTCAACATCGATGTAATGCCTACTGCCTCGGAin SEQ ID NO 12 or the corresponding regions of SEQ ID NOs. 6, 7, 13 or14.

In one embodiment, the inhibitory nucleic acid is an siRNA or an shRNAcomprising 19 to 29 nucleotides that are substantially complementary toa sequence of 19 to 29 nucleotides of Exosc10 (SEQ ID NO. 8 or 15),specifically in the region of nucleotides 340-401, 203 to 221, or1624-1642 of SEQ ID No. 15, and the corresponding regions of SEQ ID NO.8. Specific regions within 340-401 include 340-358 and 383-401.

In one aspect, siRNAs to Exosc10 (SEQ ID NO. 15) includeGAAGUAAAGUGACUGAAUU (nucleotides 340-358, SEQ ID NO. 29),CGAUACCAAUGACGUGAUA (nucleotides 383-401, SEQ ID NO. 30),ACAGUUCGGUGACGAGUAU (nucleotides 203-221, SEQ ID NO. 31),ACGGAUAUGUUCUACCAAA (nucleotides 1624-1642, SEQ ID NO. 32), thecorresponding nucleotides of SEQ ID NO. 8, and combinations thereof.

As used herein, the term substantially complementary means that thecomplement of one molecule is substantially identical to the othermolecule. Two nucleic acid or protein sequences are consideredsubstantially identical if, when optimally aligned, they share at leastabout 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% identical. Optimal alignment of sequences forcomparisons of identity may be conducted using a variety of algorithmsknown in the art. Sequence identity may also be determined using theBLAST algorithm.

Also included herein is a composition comprising a small interferingRNA, the small interfering RNA comprising 19 to 29 nucleotides that aresubstantially complementary to a sequence of 19 to 29 nucleotides ofExosc8, Exosc9, Dis3, Dis3L or Exosc10. In one aspect, the smallinterfering nucleic acid comprises 19 to 29 nucleotides that aresubstantially complementary to a sequence of 19 to 29 nucleotides ofExosc8 (SEQ ID NO. 1 or 9). In another aspect, the small interferingnucleic acid comprises 19 to 29 nucleotides that are substantiallycomplementary to a sequence of 19 to 29 nucleotides of Exosc9 (SEQ IDNO. 2, 3 or 10). In another aspect, the small interfering nucleic acidcomprises 19 to 29 nucleotides that are substantially complementary to asequence of 19 to 29 nucleotides of Dis3 (SEQ ID NO. 4, 5 or 11). Inanother aspect, the small interfering nucleic acid comprises 19 to 29nucleotides that are substantially complementary to a sequence of 19 to29 nucleotides of Dis3L (SEQ ID NO. 6, 7, 12, 13 or 14). In anotheraspect, the small interfering nucleic acid comprises 19 to 29nucleotides that are substantially complementary to a sequence of 19 to29 nucleotides of Exosc10 (SEQ ID NO. 8 or 15). Exemplary smallinterfering RNAs are siRNA and shRNA.

Further included are pharmaceutical compositions comprising apharmaceutically acceptable excipient and a small interfering RNA, thesmall interfering RNA comprising 19 to 29 nucleotides that aresubstantially complementary to a sequence of 19 to 29 nucleotides ofExosc8, Exosc9, Dis3, Dis3L or Exosc10. In one aspect, the smallinterfering nucleic acid comprises 19 to 29 nucleotides that aresubstantially complementary to a sequence of 19 to 29 nucleotides ofExosc8 (SEQ ID NO. 1 or 9). In another aspect, the small interferingnucleic acid comprises 19 to 29 nucleotides that are substantiallycomplementary to a sequence of 19 to 29 nucleotides of Exosc9 (SEQ IDNO. 2, 3 or 10). In another aspect, the small interfering nucleic acidcomprises 19 to 29 nucleotides that are substantially complementary to asequence of 19 to 29 nucleotides of Dis3 (SEQ ID NO. 4, 5 or 11). Inanother aspect, the small interfering nucleic acid comprises 19 to 29nucleotides that are substantially complementary to a sequence of 19 to29 nucleotides of Dis3L (SEQ ID NO. 6, 7, 12, 13 or 14) In anotheraspect, the small interfering nucleic acid comprises 19 to 29nucleotides that are substantially complementary to a sequence of 19 to29 nucleotides of Exosc10 (SEQ ID NO. 8 or 15). Exemplary smallinterfering RNAs are siRNA and shRNA including the moleculesspecifically described herein, or the corresponding sequences in thehuman Exosc8, Exosc9, Dis3, Dis3L and Exosc10.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Methods

Cell culture: G1E-ER-GATA-1 cells were cultured in Iscove's ModifiedDulbecco's Medium (IMDM) (Gibco®) containing 15% FBS (Gemini BioProducts), 1% penicillin-streptomycin (Cellgro®), 2 U/ml erythropoietin,120 nM monothioglycerol (Sigma-Aldrich®), 0.6% conditioned medium from akit ligand producing CHO cell line, and 1 μg/ml puromycin (Gemini BioProducts). Cells were treated with 1 μM β-estradiol (Steraloids, Inc) toinduce ER-GATA-1 activity. G1E cells were cultured in the same media asG1E-ER-GATA-1 cells without puromycin. Primary fetal liver erythroidprogenitor cells were cultured in StemPro®-34 (Gibco®) supplemented with10% nutrient supplement (Gibco), 2 mM L-glutamine (Cellgro®), 1%penicillin-streptomycin (Cellgro®), 100 μM monothioglycerol(Sigma-Aldrich®), 1 μM dexamethasone (Sigma-Aldrich®), 0.5 U/ml oferythropoietin, and 1% conditioned medium from a kit ligand producingCHO cell line for expansion. For differentiation, cells were cultured inES IMDM (glutamine-free) (Hyclone®) containing 10% FBS (Gemini BioProducts), 10% PDS (Animal Technologies), 5% PFHM II (Gibco®), 2 mML-glutamine (Cellgro®), 1% penicillin-streptomycin (Cellgro®), 100 μMmonothioglycerol (Sigma-Aldrich®), and 5 U/ml of erythropoietin. Cellswere grown in a humidified incubator at 37° C. with 5% carbon dioxide.All percentages are vol/vol unless otherwise noted.

Transmission electron microscopy (TEM). Cells were fixed in 0.1M sodiumcacodylate buffer, pH 7.4, containing 2.5% glutaraldehyde and 2%paraformaldehyde overnight at 4° C. The cells were then post-fixed in 2%Osmium Tetroxide in 0.1 M sodium cacodylate for 2 h at room temperature.Following post-fixation, the samples were dehydrated in a graded ethanolseries, then further dehydrated in propylene oxide and embedded in Eponepoxy resin. Samples were sectioned for TEM using a Reichert-JungUltracut-E Ultramicrotome and contrasted with Reynolds lead citrate and8% uranyl acetate in 50% EtOH. Ultrathin sections were observed with aPhilips CM120 electron microscope, and images were captured with aMegaView III side mounted digital camera.

TEM immunogold staining. Cells were washed with PBS and fixed in 0.1 MPB buffer (0.2 M sodium phosphate monobasic combined with 0.2 M sodiumphosphate dibasic, pH 7.4) containing 0.1% glutaraldehyde and 4%paraformaldehyde overnight at 4° C. Fixed cells were embedded in 3%agarose, which was sectioned using a vibratome. After washing with 0.1 MPB buffer, freshly prepared 0.1% NaBH₄ in 0.1 M PB was used toinactivate residual aldehyde groups for 10 min. Following rinsing with0.1 M PB buffer, specimens were permeabilized in PBS containing 0.2%Triton X-100 for 30 min at room temperature with gentle agitation,followed by PBS washes. AURION blocking solution was used to block for 1h at room temperature. After washing with incubation buffer (0.2% AURIONBSA-c in PBS), specimens were incubated with LC3 rabbit polyclonalantibody (PM036, MBL) overnight at 4° C., followed by extensive washingwith incubation buffer. Samples were incubated with ultra-smallgold-conjugated secondary antibody (1/100 dilution) in incubation bufferovernight at 4° C. Following extensive washing with incubation buffer,specimens were washed with PBS before post-fixation with 0.1 M PBcontaining 2% glutaraldehyde for 1 h at room temperature. After washingwith 0.1 M PB followed by ECS solution, specimens were incubated withsilver enhancement solution for 2 h, washed extensively with ECSsolution, dehydrated, and embedded following the TEM procedure describedabove.

Protein analysis. Equal numbers of cells were boiled for 10 min in SDSsample buffer (25 mM Tris, pH 6.8, 2% β-mercaptoethanol, 3% SDS, 0.005%bromophenol blue, and 5% glycerol). Samples were resolved by SDS-PAGE,and proteins were measured by semi-quantitative Western blotting withECL Plus™ (Pierce®). GATA-1 rat monoclonal antibody (SC-265) and Foxo3rabbit polyclonal antibody (07-702) were from Santa Cruz Biotechnologyand Millipore, respectively. β-actin mouse monoclonal antibody (3700)was from Cell Signaling Technology, and LC3B mouse monoclonal antibody(M115-3) (Medical and Biological Laboratories) were used.

Quantitative real-time RT-PCR. Total RNA was purified with Trizol®(Invitrogen™). cDNA was prepared by annealing 1.5 μg of RNA with 250 ngof a 5:1 mixture of random hexamer and oligo (dT) primers heated at 68°C. for 10 min. This was followed by incubation with Murine MoloneyLeukemia Virus Reverse Transcriptase (Invitrogen™) combined with 10 mMDTT, RNasin (Promega), and 0.5 mM dNTPs at 42° C. for 1 h. The mixturewas diluted to a final volume of 120 μl and heat inactivated at 95° C.for 5 min RT-PCR reactions (20 μl) contained 2 μl of cDNA, appropriateprimers, and 10 μl of SYBR green master mix (Applied Biosystems®).Product accumulation was monitored by SYBR green fluorescence. Astandard curve of serial dilutions of cDNA samples was used to determinerelative expression. mRNA levels were normalized to 18S rRNA.

Quantitative chromatin immunoprecipitation (ChIP) assay. ChIP wasconducted as known in the prior art. ER-GATA-1 and endogenous GATA-1were immunoprecipitated with rabbit anti-GATA-1 antibody, and Foxo3 wasimmunoprecipitated with rabbit anti-Foxo3 antibodies (Millipore andSanta Cruz Biotechnology). Rabbit preimmune sera (PI, Covance) was usedas a control. Phosphorylated RNA Polymerase II was immunoprecipitatedwith mouse anti-pSer5 Pol II antibody (Covance). Mouse IgMp. (Sigma) wasused as a control. Samples were analyzed by quantitative real-time PCR(ABI StepOnePlus™), and product was measured by SYBR Green fluorescence.The amount of product was determined relative to a standard curvegenerated from a serial dilution of input chromatin. Dissociation curvesrevealed that primer pairs generated single products.

Primary murine bone marrow erythroid precursors. Murine bone marrowerythroblasts were isolated from femur and tibia from 8 week old mice.Bone marrow cells were resuspended in 90 μl of MACS buffer (PBScontaining 0.5% BSA and 2 mM EDTA) per 5×10⁶ cells and 10 μl ofAnti-Ter119 Microbeads (Miltenyi Biotec) were added. After 15 minincubation at 4° C., a 15 fold volume of MACS buffer was added to washmicrobead-cell complexes. After centrifugation at 1200 rpm for 10 min at4° C., cells were resuspended in 1 ml MACS buffer per 1×10⁸ cells andapplied to the MACS LS column. After washing the column four times with3 ml of MACS buffer, Ter119 microbead-conjugated cells were eluted twicewith 2.5 ml MACS buffer.

Primary fetal liver erythroid progenitor cell isolation. Embryonic day14.5 fetal livers were used to isolate primary erythroid progenitorcells using EasySep™ negative selection Mouse Hematopoietic ProgenitorCell Enrichment Kit (StemCell™ Technologies). Briefly, fetal liver cellswere resuspended at a concentration of 5×10⁷ cells/ml in PBS containing2% FBS, 2.5 mM EDTA, and 10 mM glucose, and EasySep™ Mouse HematopoieticProgenitor Cell Enrichment Cocktail was added at 50 μl/ml supplementedwith 2.5 μg/ml biotin-conjugated CD71 antibody (eBioscience). After 15min incubation on ice, cells were washed once by centrifugation for 5min at 1200 rpm at 4° C. Cells were resuspended at a concentration of5×10⁷ cells/nil in PBS containing 2% FBS, 2.5 mM EDTA, and 10 mMglucose, and EasySep™ Biotin Selection Cocktail was added at 100 μl/ml.After 15 min on ice, EasySep™ Mouse Progenitor Magnetic Microparticleswere added at 50 μl/ml. After 10 min on ice, cells were resuspended to atotal volume of 4 ml and incubated with a magnet for 3 min. Unboundprogenitor cells were transferred into a 15 ml tube and used forsubsequent experiments.

siRNA-mediated knockdown. Dharmacon siGENOME® Smartpools against mouseFoxo3, Exosc8, Exosc10, Dis3, Dis3l were used with non-targeting siRNApool as a control. siRNA (240 pmol) was transfected into 3×10⁶ ofG1E-ER-GATA-1 cells or G1E cells using the Amaxa® Nucleofector™ Kit R(Amaxa® Inc.) with program G-16. siRNA was transfected twice at 0 and 24h. G1E-ER-GATA-1 cells were treated with β-estradiol for the indicatedtimes. The smartpool for Exosc8 includes GAAAGAGGAUUUAUGCAUU(nucleotides 712-730, SEQ ID NO. 16), CAACAUAGGUUCAAUCAGU (nucleotides451-469, SEQ ID NO. 17), UGGAGCCGCUGGAGUAUUA (nucleotides 363-381, SEQID NO. 18), and GGAAUACCACGGUCAUUUG (nucleotides 501-519, SEQ ID NO.19). The smartpool for Exosc10 includes GAAGUAAAGUGACUGAAUU (nucleotides340-358, SEQ ID NO. 29), CGAUACCAAUGACGUGAUA (nucleotides 383-401, SEQID NO. 30), ACAGUUCGGUGACGAGUAU (nucleotides 203-221, SEQ ID NO. 31),and ACGGAUAUGUUCUACCAAA (nucleotides 1624-1642, SEQ ID NO. 32).

shRNA-mediated knockdown. MiR-30 context Exosc8 (Rrp43), Dis3 (Rrp6),Dis3L and Exosc9 (Rrp45) shRNAs were cloned into MSCV-PIG vector kindlyprovided by Dr. Mitchell Weiss using BglII and XhoI sites. 1×10⁵ primaryerythroid progenitor cells were spinfected with 100 μl of retrovirussupernatant and 8 μg/ml polybrene in 400 μl of fetal liver expansionmedia at 1200×g for 90 min at 30° C.

     

MiR-30 context Exosc8 shRNA 1 sequence: (SEQ ID NO. 20)TGCTGTTGACAGTGAGCGCCAGGAGATTTCTGAAGGAAAATAGTGAAGCCACAGATGATTTTCCTTCAGAAATCTCCTGTTGCCTACTGCCTCGGA.MiR-30 context Exosc8 shRNA 2 sequence: (SEQ ID NO. 21)TGCTGTTGACAGTGAGCGACGCTGGAGTATTACAGGAGATTAGTGAAGCCACAGATGTAATCTCCTGTAATACTCCAGCGGTGCCTACTGCCTCGGA.MiR-30 context Exosc9 shRNA 1 sequence: (SEQ ID NO. 23)TGCTGTTGACAGTGAGCGACAGATTGGAGACGGAATAGAATAGTGAAGCCACAGATGTATTCTATTCCGTCTCCAATCTGGTGCCTACTGCCTCGGAMiR-30 context Exosc9 shRNA 2 sequence: (SEQ ID NO. 24)TGCTGTTGACAGTGAGCGAAAGAAGAGAACTGCTAACTAATAGTGAAGCCACAGATGTATTAGTTAGCAGTTCTCTTCTTCTGCCTACTGCCTCGGA.MiR-30 context Dis3 shRNA 1 sequence (SEQ ID No. 25):TGCTGTTGACAGTGAGCGCCCACAGATCCCAGGAATAAATTAGTGAAGCCACAGATGTAATTTATTCCTGGGATCTGTGGTTGCCTACTGCCTCGGAMiR-30 context Dis3 shRNA 2 sequence (SEQ ID No. 26):TGCTGTTGACAGTGAGCGACAGACAGTCAGCTGCAAGTTATAGTGAAGCCACAGATGTATAACTTGCAGCTGACTGTCTGCTGCCTACTGCCTCGGAMiR-30 context Dis3L shRNA 1 sequence (SEQ ID No. 27):TGCTGTTGACAGTGAGCGCGGCCGGAGACAGTATAACAAATAGTGAAGCCACAGATGTATTTGTTATACTGTCTCCGGCCTTGCCTACTGCCTCGGAMiR-30 context Dis3 shRNA 1 sequence (SEQ ID No. 28):TGCTGTTGACAGTGAGCGCTACATCGATGTTGAAGCTAGATAGTGAAGCCACAGATGTATCTAGCTTCAACATCGATGTAATGCCTACTGCCTCGGABold sequences denote sense and antisense sequences.

Flow cytometry. Cells were washed with PBS once, and 1×10⁶ cells werestained with 0.8 μg of anti-mouse Ter119-APC and anti-mouse CD71-PE(eBioscience) at 4° C. for 30 min in the dark. After staining, cellswere washed three times with 2% BSA in PBS. For Exosc8 knockdownexperiments, samples were analyzed using a BD LSR II flow cytometer (BDBiosciences). For Exosc9 knockdown experiments, which included an Exosc8knockdown positive control, Ten 19 and CD71 staining was analyzed usinga BD FACSAria II cell sorter (BD Biosciences), at which time shRNAexpressing cells were sorted from the total population, utilizing theGFP marker which is co-expressed with the shRNA, for subsequenttranscriptional analysis. DAPI (Sigma) staining discriminated deadcells.

Cell cycle analysis. Cells were resuspended at 5×10⁵ cells/nil in mediumcontaining 20 μg/ml Hoechst 33342 (Invitrogen) and incubated at 37° C.for 30 min. After incubation, this cell suspension was adjusted to 2×10⁶cells/ml. DNA contents were measured using BD™ LSR II (BD™ Biosciences)and Modfit LT 3.2.1 (Verity software house) was used to analyze thedata.

Transcriptional profiling. Amino Allyl RNA was synthesized from mRNA,labeled, and hybridized to 8×60K Mouse Whole Genome arrays (Agilent)with three biological replicates. Arrays were read utilizing a G-2505CDNA Microarray Scanner with Surescan High Resolution (Agilent). EDGE3web-based two-color microarray analysis software and Microsoft Excelwere used for data analysis.

Immunofluorescence. To detect endogenous LC3B, cytospun cells were fixedwith 3.7% paraformaldehyde in PBS for 10 min at room temperature. Afterwashing with PBS, cells were permeabilized using 0.2% Triton X-100 inPBS for 15 min at room temperature. After washing with PBS, slides wereblocked with 10% blocking solution BlokHen® (Ayes Labs, Inc.) in PBS-Tfor 1 h at 37° C. and then incubated with 1.6 μg/ml LC3 rabbitpolyclonal antibody (Medical and Biological Laboratories) in 2% BlokHen®in PBS-T overnight at 4° C. After washing three times with PBS-T, slideswere incubated with 8 μg/ml Alexa Fluor 488 goat anti-rabbit IgGantibody (Invitrogen) for 1 h at 37° C. Slides were washed three timeswith PBS-T and mounted using Vectashield® mounting medium with DAPI(Vector Laboratories, Inc.). Cells were photographed using a 100×/1.4oil objective (Olympus IX2-UCB). For quantitation, cells containing morethan two LC3-positive punctae structures were scored asautophagosome-positive cells. At least 200 cells were counted per slide,and three independent biological replicates were counted.

Generation of 3D exosome structure: Protein structure coordinate filesfor the human exosome complex were downloaded from the ResearchCollaboratory for Structural Bioinformatics Protein Data Bank (accessionnumber 2NN6). Structural images were generated using PyMOL.

Statistical analysis. Paired Student's T-test with the web-based toolwas used to calculate statistical significance. EDGE3 analysis softwareand online NIH DAVID tool were used for statistical analysis ofmicroarray data and gene ontology (GO) terms analysis, respectively.

Example 1 Autophagy Induction Via GATA-1/Foxo3 Feed-Forward Loop

To gain mechanistic insights into how autophagy is initiated in aspecific developmental context, a genetic complementation analysis wasconducted in GATA-1-null G1E-ER-GATA-1 cells that stably expressER-GATA-1 (subsequently referred to as GATA-1). Conditional GATA-1activation with estradiol induces a physiological erythroid geneexpression program and erythroid maturation, recapitulating a window ofthe maturation process that occurs in vivo. GATA-1 directly inducesselect autophagy genes and MAP1LC3B (LC3B)-positive punctae.Transmission electron microscopy (TEM) was used to test whetherGATA-1-induced punctae detected by immunofluorescence analysis representautophagosomes/autolysosomes. GATA-1 activation for 24 or 48 hoursinduced erythroid maturation associated with accumulation of largecytoplasmic vesicles (FIG. 1). Both the number of vesicles per cell andvesicle size increased, although mature cells retained some smallvesicles (<0.5 μm²) (data not shown). Immunogold TEM analysis withanti-LC3B antibody confirmed that GATA-1-induced vesicles contain theautophagosome marker LC3B and bear morphological attributes ofautophagosomes/autolysosomes (data not shown). Thus, GATA-1 increasesthe number and size of these autophagy structures as an important stepin erythroid cell maturation.

It has been shown that as siRNA-mediated downregulation of Foxo3decreased GATA-1-induced expression of select autophagy genes, withoutaffecting all GATA-1 target genes. Foxo3 amplifies GATA-1 activity in acontext-dependent manner. To test the implications of this Foxo3activity for autophagy gene expression in vivo, autophagy geneexpression in primary bone marrow Ter119+ cells from wild type andFoxo3^(−/−) mice was quantitated. Foxo3 mRNA was undetectable, and Gata1mRNA was approximately 30% higher in Ten 19+ cells from Foxo3^(−/−)versus Foxo3^(+/+) bone marrow (data not shown). Foxo3 and GATA-1protein levels were concordant with mRNA levels (data not shown). Theexpression of autophagy genes regulated by GATA-1 in G1E-ER-GATA-1 cells(Map1lc3b, Gabarapl1, Gate-16) decreased significantly by 50-60% (FIG.2). Thus, Foxo3 amplifies expression of these genes in vivo, validatingthe G1E-ER-GATA-1 results.

To elucidate how GATA-1 and Foxo3 function combinatorially to regulateautophagy genes, it was determined whether they co-occupy chromatin atautophagy gene loci. Using a quantitative ChIP assay, GATA-1 and Foxo3occupancy was measured at chromatin sites containing Foxo3 DNA motifs.GATA-1 and Foxo3 occupied indistinguishable sites at Gabarapl1 andGate-16 (data not shown). To test whether endogenous GATA-1 and Foxo3co-occupy chromatin in primary erythroblasts, quantitative ChIP analysiswas conducted with primary Ter119+ cells from mouse bone marrow. GATA-1and Foxo3 co-occupied sites at Gabarapl1 and Gate-16, recapitulating theG1E-ER-GATA-1 results (data not shown). GATA-1 and FOX03 co-occupancy atGABARAPL1 and GATE-16 was also confirmed in primary erythroblastsderived from human CD34+ peripheral blood mononuclear cells (data notshown). The co-occupancy might reflect the presence of both factors in acomplex at an identical chromatin site or independent occupancy ofneighboring sites that cannot be segregated due to resolutionlimitations (data not shown). To test these models, tiled primers wereused to quantitate GATA-1 and Foxo3 occupancy over a broader chromosomalregion (data not shown). Maximal GATA-1 and Foxo3 occupancy occurred atdistinct sites containing GATA and Foxo3 DNA motifs respectively,suggesting that GATA-1 and Foxo3 occupy neighboring sites (data notshown).

Given the close proximity of the GATA-1 and Foxo3 occupancy sites, itwas investigated whether GATA-1 regulates Foxo3 chromatin occupancy andvice versa. GATA-1 induced Foxo3 chromatin occupancy at select autophagyloci and at additional GATA-1 target genes (<3 fold, p<0.0001), but notat all GATA-1 target genes (e.g., at Lyl1 promoter and Gata2-2.8 site)in G1E-ER-GATA-1 cells (FIG. 3). The Alas2-4.7 kb site, which was notbound by GATA-1, served as a negative control. It was also determinedwhether Foxo3 increases GATA-1 chromatin occupancy. Knocking-down Foxo3expression (data not shown) increased GATA-1 occupancy up to 30% atautophagy and non-autophagy gene loci (data not shown). Thus, whileGATA-1 induced Foxo3 chromatin occupancy, downregulating Foxo3 did notdecrease GATA-1 chromatin occupancy. As suggested previously that GATA-1induces Foxo3 mRNA and occupies the FOX03 locus, GATA-1 induced Foxo3protein 6.7 fold (p<0.001) (data not shown). These results illustrate atype I coherent feed-forward loop, in which GATA-1 directly activatesautophagy genes and induces Foxo3, and Foxo3 also activates theautophagy genes (FIG. 4). The model predicts that the feed-forward loopwould buffer against transiently elevated input stimuli (e.g., activeGATA-1 and/or Foxo3), thereby suppressing the premature induction ofautophagy, which would be deleterious to the erythroblast.

Example 2 GATA-1/Foxo3 Cooperativity in Erythroid Cell Biology

To determine the extent of the GATA-1/Foxo3-regulated genetic network inerythroid cells, the transcriptome of Foxo3-knockdown versus controlcells was profiled in the context of active GATA-1. Comparison of theFoxo3-regulated genes to the existing GATA-1 target gene dataset inG1E-ER-GATA-1 cells revealed 498 of the 2006 Foxo3-regulated genes(approximately 25%) as GATA-1-regulated. GATA-1/Foxo3 co-regulate acohort of genes in erythroid cells more extensive than solely theautophagy genes (FIG. 5). The co-regulated genes were classified asGATA-1- or Foxo3-activated or -repressed. GATA-1 and Foxo3 regulated 78%of the co-regulated genes in the same direction (FIG. 6). GATA-1/Foxo3activated and repressed 235 and 154 genes, respectively (FIG. 6). Geneontology (GO) analysis was conducted to assess whether these distinctregulatory modes have hallmark functional signatures, and the top fiveGO terms ascribed to the co-activated genes implicate apoptosis andautophagy (FIG. 7). Genes involved in “non-coding RNA metabolic process”or “RNA processing” were uniquely and significantly enriched in theco-repressed cohort (FIG. 8). Genes assigned to these categories arelisted Table 1A and B. Importantly, these genes could not have beenpredicted from prior genomic analyses of GATA factor function orhematopoiesis.

TABLE 1A Regulation of Apoptosis Gene Fold Change Name GATA-1 Foxo3 Atf51.81 2.85 B4galt1 4.49 2.61 Cdkn1b 6.59 2.33 Cln8 7.43 1.88 Ddit3 4.391.88 Dedd2 3.13 1.90 Il4 13.6 1.88 Rb1cc1 2.49 2.56 Serinc3 3.84 2.39Sgms1 2.58 1.89 Ube2b 1.87 2.11 Tax1bp1 2.24 1.75 Trp53inp1 10.7 3.73

TABLE 1B ncRNA Metabolic Process Gene Fold Change Name GATA-1 Foxo3Aarsd1 −2.10 −1.71 Cars2 −3.51 −1.71 Exosc5 −2.07 −1.77 Exosc8 −1.74−2.40 Lin28b −2.97 −2.19 Naf1 −3.85 −1.71 Nop10 −1.78 −1.57 Nsa2 −2.27−2.08 Nsun2 −3.51 −1.73 Rpp38 −2.57 −1.80 Tarsl2 −1.85 −2.68 Tsen2 −2.46−2.06 Wdr4 −3.67 −2.05 Wdr36 −2.38 −1.64 Wdr55 −2.19 −1.57

Example 3 Linking the GATA-1/Foxo3 Feed-Forward Loop to RNA SurveillanceMachinery

Two of the highly GATA-1/Foxo3-repressed genes implicated in “non-codingRNA metabolic process” were Exosc8 and Exosc5 (Rrp46), essential corecomponents of the exosome complex. Exosc8 contains a catalyticallyinactive RNase PH domain Using real-time RT-PCR to confirm themicroarray data, GATA-1 repressed Exosc8 expression 25 fold, andknocking-down Foxo3 de-repressed Exosc8 expression 3 fold (FIGS. 9 and10). Endogenous GATA-1 occupied the EXOSC8 promoter in primary humanerythroblasts, suggesting direct GATA-1 transcriptional regulation (FIG.11). The kinetics of GATA-1-mediated Exosc8 repression are slower thanprototypical GATA-1-repressed target genes and resemble the slowinduction of the direct GATA-1 target βmajor, which has been attributedto a GATA-1 requirement to upregulate FOG-1 expression. GATA-1 would berequired to upregulate Foxo3 as a prelude to functioning combinatoriallywith Foxo3 to confer maximal Exosc8 repression. This method of generegulation delays the reversal of the response (Exosc8 repression) uponloss of the activating stimuli (GATA-1 and Foxo3).

Example 4 Exosc8 Regulates a Cohort of GATA-1 Target Genes Essential forErythroid Maturation

Though GATA-1 represses as many genes as it activates, the functionalsignificance of many of the GATA-1-repressed genes is not established.Without being held to theory, it is believed that a cohort of theencoded proteins promote precursor cell expansion or survival, thusactively counteracting maturation. Since GATA factors have not beenlinked to the exosome complex, the functional consequences ofGATA-1-mediated Exosc8 repression were investigated by knocking-downExosc8 in G1E-ER-GATA-1 cells. (FIG. 12) Knocking down Exosc8 byapproximately 80% significantly enhanced expression of severalGATA-1-activated genes by 3 to 12-fold (βmajor, Alas2, and Slc4a1),while other GATA-1 activated genes (Epb4.9 and Fog-1) were largelyunaffected. GATA-1-mediated repression of Gata2 and Lyl1 were unalteredby the knockdown (FIG. 13).

In principle, impairing the mRNA degradation function of the exosomecomplex by disturbing a core subunit could lead to the accumulation ofmRNAs. To determine whether the gene expression alterations upon Exosc8downregulation resulted solely from altered mRNA degradation, primaryunprocessed transcripts were quantitated for Exosc8-responsive,GATA-1-activated genes, using the Exosc8-unresponsive gene Epb4.9 as acontrol. Similar to the mRNA analysis, βmajor, Alas2, and Slc4a1 primarytranscripts increased 2 to 7-fold, while Epb4.9 primary transcripts werelargely insensitive (FIG. 14) Additionally the expression of the geneencoding the large subunit of RNA polymerase II (RPH215) and anautophagy gene that is not GATA-1-regulated, Atg16l1 was measured.RPII215 and Atg16l1 expression levels were unaffected (data not shown).Thus, Exosc8 suppression of a select cohort of erythroid mRNAs does notinvolve an exclusive mRNA degradation mechanism.

Example 5 The Exosome Complex as an Endogenous Suppressor of Autophagy

Since autophagy genes are GATA-1/Foxo3-activated and mediate animportant aspect of erythroid maturation, it was hypothesized thatGATA-1/Foxo3-repressed genes may also be essential for the maturationprogram. Knocking-down Exosc8 by approximately 80% significantlyupregulated Map1lc3b and Gate-16 expression approximately 2-fold (FIG.15) and upregulated the active, lipid-conjugated form of LC3B (LC3-II),even without GATA-1 activity (FIG. 16). Since LC3-II is a hallmark ofautophagy, it was tested whether knocking-down Exosc8 inducesautophagosome accumulation. Consistent with the LC3B upregulationdetected by Western blotting, immunofluorescence analysis indicated thatknocking-down Exosc8 significantly induced autophagosome accumulation incells lacking or containing active GATA-1 (data not shown and FIG. 17).The number of autophagosomes per cell was higher in Exosc8-knockdowncells lacking GATA-1 activity (FIG. 17). As the Exosc8 knockdown inducedautophagy, these results suggest that GATA-1-dependent Exosc8 repressionin a physiological context instigates and/or enhances autophagy duringerythroid cell maturation.

Example 6 Exosome Complex Structure and Expression During PrimaryErythroblast Maturation

The exosome complex consists of 9 core subunits (Exosc1-9). Exosc1(Csl4), Exosc2 (Rrp4) and Exosc3 (Rrp40) are RNA binding proteins.Biochemical analyses demonstrate that Exosc4/Exosc9 (Rrp41/Rrp45),Exosc6/Exosc7 (Mtr3/Rrp42) and yeast, but not human, Exosc5/Exosc8(Rrp46/Rrp43) form stable dimers. Human EXOSC6/EXOSC7/EXOSC8 form astable trimer (FIG. 18). Exosc1 interacts with Exosc6, Exosc2 interactswith both Exosc4 and Exosc7, and Exosc3 interacts with both Exosc5 andExosc9. Thus, the RNA binding proteins stabilize subunit interactionswithin the complex (FIG. 18). Three catalytic subunits, Exosc10 (Rrp6),Dis3 (Rrp44) and Dis3l (a human homologue of yeast Dis3, not found inyeast) can bind to the core exosome. Exosc10 and Dis3l are 3′ to 5′exoribonucleases, while Dis3 exhibits both endoribonuclease andexoribonuclease activity. Dis3 is mainly associated with the nuclearexosome complex and is involved in the degradation of rRNA and PROMTs(promoter upstream transcripts). Dis3l localizes in the cytoplasm whereit mediates selective mRNA degradation. Exosc10 increases the stabilityof the exosome complex and interacts directly with Exosc1, Exosc6, andExosc8 (FIG. 18). Exosc10 improves the ability of the exosome complex toprocess substrates using the long RNA-binding pathway by positioningExosc1 correctly to bind RNA. Exosc10 localizes predominantly in thenucleus and is highly enriched in the nucleolus, indicating a role inpre-rRNA processing, although a small percentage is cytoplasmic. Dis3 orDis3l binds to the exosome core opposite of Exosc10. The Dis3 (or Dis3l)N-terminus wedges between Exosc4 and Exosc7 and approaches the Exosc2N-terminus (FIG. 18).

To assess whether Exosc8 expression in G1E-ER-GATA-1 cells reflects itsbehavior upon primary erythroblast maturation, genomic databases weremined and the expression pattern of the exosome components in primaryerythroblasts were determined. During definitive erythropoiesis in themouse (FIG. 19) and human (FIG. 20), expression of exosome complexcomponents, including Exosc8, decreased. The direct repression of Exosc8by GATA-1/Foxo3, and Exosc8 repression upon murine and human erythroidcell maturation suggest important functional implications of the exosomecomplex in erythroid cell biology.

Example 7 Exosc8 Suppresses Primary Erythroblast Maturation

It was further tested whether Exosc8 suppresses a cohort oferythroid-specific RNAs in primary murine erythroid precursor cellsisolated from embryonic day 14.5 fetal livers. Freshly isolated cellswere infected with two distinct Exosc8 shRNA-expressing retroviruses,expanded for 3 days, and differentiated for 1 day. shRNA targetingluciferase was used as a control. The two Exosc8 shRNAs inducedqualitatively similar results. Consistent with the G1E-ER-GATA-1 cellresults, Exosc8 shRNA upregulated βmajor, Alas2, and Slc4a1 expression,while GA TA-1 and Fog-1 expression were largely unaffected duringexpansion culture conditions (FIG. 21). The expression of thetranscription factor PU.1, which can oppose GATA-1 activity in certaincontexts, was significantly decreased in the Exosc8-knockdown cellsunder expansion conditions (FIG. 21). These data suggest thatGATA-1-mediated repression of Exosc8 during erythropoiesis contributesto activation of GATA-1 target genes important for erythroid maturation.

Consistent with the upregulation of erythroid-specific genes, Exosc8knockdown cells were considerably smaller and red, indicatinghemoglobinization, versus control cells, even when differentiation wasnot induced. The maturation status of control and Exosc8-knockdown cellswere assessed using flow cytometric quantitation of erythroid surfacemarkers Ten 19 and CD71 and Wright-Giemsa staining. Based on CD71 andTer119 expression during expansion culture conditions, most cellsrepresented R2 (proerythroblasts and early basophilic erythroblasts) andR3 (early and late basophilic erythroblasts) populations in the control(FIG. 22). The Exosc8 knockdown induced a major shift from R2 to R3 andR4 (polychromatophilic and orthochromatic erythroblasts) and even to R5(late orthochromatic erythroblasts and reticulocytes) populations,without influencing cell viability, in every condition tested (FIG. 22).Consistent with enhanced erythroid maturation, the Exosc8-knockdowncells exhibited hallmark morphological features of mature erythroblastsand reticulocytes. The knockdown induced accumulation of cells with morecondensed nuclei, and the cytoplasmic color became slate blue indicatinghemoglobinization (FIG. 23). Forward scatter (FSC) measurementsconfirmed that the Exosc8-knockdown cells were significantly smallerthan control cells (FIG. 23).

Similar to G1E-ER-GATA-1 cells, knocking-down Exosc8 increasedautophagosome-positive erythroid precursor cells 13 fold in theexpansion stage and 2 fold upon differentiation (FIG. 24). The number ofautophagosomes per positive cell was similar in all conditions (FIG.24). Thus, Exosc8 suppresses autophagy in primary erythroid precursorcells.

During expansion of the primary erythroblasts, it was observed thatExosc8-knockdown samples consistently contained fewer cells than thecontrol samples, leading to the analysis of their cell cycle status.G₀/G₁ populations increased approximately 2-fold, while S and G₂/M phasecells decreased approximately 2-fold, indicating that the Exosc8knockdown induced cell cycle exit, consistent with expectations forerythroid cell maturation. (FIG. 25).

The Exosc8 knockdown would be expected to impair exosome complexactivity to degrade certain mRNAs, thereby post-transcriptionallyupregulating mRNAs. However, the Exosc8 knockdown also increased primarytranscripts (FIGS. 14 and 15), suggesting a mechanism not restricted tocytoplasmic mRNA degradation. The possibility that elevated primarytranscripts resulting from Exosc8 knockdown may reflect increasedtranscription of the respective genes was explored. In this regard, theexosome complex can function via epigenetic regulation and non-codingRNA surveillance, which impact transcription. It was tested whetherExosc8 regulates serine 5-phosphorylated RNA Polymerase II (pSer5-PolII) occupancy at the affected promoters. Quantitative ChIP analysis inprimary fetal liver erythroid progenitors revealed that the Exosc8knockdown elevated pSer5-Pol II occupancy at βmajor, Alas2 and Slc4a1promoters, but not at the active Eif3k promoter and the repressed Necdinpromoter, indicating that Exosc8 represses transcription of theseerythroid genes (FIG. 26). In aggregate, these data demonstrate thatExosc8 suppresses maturation of primary erythroid precursor cells.

Example 8 The Exosome Complex as an Endogenous Suppressor of ErythroidCell Maturation

It was considered whether Exosc8 suppresses erythroid maturation byfunctioning within the exosome complex or independent of the exosomecomplex. To distinguish between these models, it was tested whetheranother exosome component, Exosc9, also suppresses erythroid maturation.Exosc9 was prioritized due to its integral position within the exosomecomplex, where it binds Exosc4 and interacts with Exosc8 via itsC-terminal tail, which first interfaces with Exosc5 and then wrapsaround Exosc5 and Exosc8 (FIG. 27). Fetal liver-derived primaryerythroid precursor cells were infected with two distinct Exosc9shRNA-expressing retroviruses and the cells were expanded for 3 days andincluded Exosc8 shRNA as a positive control. Utilizing the GFP markerco-expressed with the shRNA, shRNA-expressing cells were sorted from thewhole population. Recapitulating the results for Exosc8, reducing Exosc9in primary erythroid precursor cells significantly increased theexpression of βmajor, Alas2, and Slc4a1 without affecting GA TA-1 andFog-1 (FIG. 28). Similar to Exosc8 knockdown, immunophenotypicalanalysis using CD71 and Ter119 staining showed a major shift from R2(proerythroblasts and early basophilic erythroblasts) to R3 (early andlate basophilic erythroblasts) and R4 (polychromatophilic andorthochromatic erythroblasts) upon Exosc9 knockdown (FIG. 29).Wright-Giemsa staining demonstrated enhanced erythroid maturation in theExosc9 knockdown cells (FIG. 30). To identify the role of the catalyticactivity of the exosome in the Exosc8/Exosc9 mediated repression oferythroid maturation we knocked down two catalytic components of theexosome, Dis3 and Dis3L. Knockdown of both Dis3 (FIG. 31) and Dis3L(FIG. 32) reduced the number of cells in the R1 population and increasedthe R3, R4 and R5 cell populations, although to a lesser degree thaneither Exosc8 or Exosc9 knockdown. The ability of Exosc9, Dis3 and Dis3Lknockdown to phenocopy the maturation effect resulting from the Exosc8knockdown suggests that Exosc8 functions within the exosome complex,and/or select components of the complex, to suppress erythroidmaturation.

To further dissect how downregulating Exosc8 promotes erythroidmaturation, the transcriptomes of Exosc8-knockdown primary murineerythroid precursor cells and control cells were compared. TheExosc8-regulated genes were also compared to either GATA-1- orFoxo3-regulated genes. To interrogate the relationship between GATA-1-and Exosc8-regulated genes, GATA-1 and Exocs8-regulated genes wereclassified as repressed or activated. The most frequent mode ofco-regulation was Exosc8-repressed and GATA-1-activated (FIG. 33). Thedifferentially regulated transcripts included those important forerythroid cell maturation, specifically heme biosynthesis (Alas2),anti-apoptosis (Bcl-xL), and the red cell cytoskeleton (Gypa). Geneontology analysis was conducted to gain functional insights into theGATA-1-activated/Exosc8-repressed genes. The top three GO terms werecell cycle arrest, regulation of apoptosis, and cellular proteincatabolic process (FIG. 34). Analysis of the Exosc8/Foxo3-regulated genecohort revealed that like the GATA-1/Exosc8-regulated genes, the mostfrequent mode of regulation was Exosc8-repressed and Foxo3-activated(FIG. 35). Gene ontology analysis indicated that Exosc8- andFoxo3-regulate genes involved in apoptosis and macromolecular metabolismin maturing erythroid cells (FIG. 36).

Given that the Exosc8 knockdown caused cells to accumulate in G₀/G₁phase, the GATA-1-activated, Exosc8-repressed genes involved in cellcycle arrest were further explored (FIG. 37). This category includedCdkn1b (p27^(kip1)), Ddit3 (CHOP), Gas2l1 (Gar22), Trp53inp1, Gadd45a,and Ern1 (FIG. 37). Cdkn1b controls the activity of cyclin-dependantkinases that bind to Cyclin D and Cyclin E, arresting cells in G₁. Ddit3(CHOP) is a DNA damage-inducible G₁/S transition inhibitor. Trp53inp1functions downstream of p53 and p73 to induce G₁ arrest and to enhanceapoptosis in multiple cell types. Ern1 is a component of the unfoldedprotein response, which is stimulated by unfolded proteins in theendoplasmic reticulum, leading to G₁ arrest. Gas2l1 (Gar22) is adownstream effector molecule of thyroid receptor and enhances Gata2 andc-Kit repression during erythroid maturation. Gas2l1 is highly expressedin cells arrested in G₁, and its overexpression in erythroid cells isassociated with a lengthened cell cycle, especially S phase. Gadd45aresponds to genotoxic stress by interacting with CDK1/Cyclin B1 toinduce G₂/M arrest; no specific role for Gadd45a in erythropoiesis hasbeen reported (FIG. 37).

RT-PCR analysis confirmed that Exosc8 knockdown in primary murineerythroid precursor cells significantly elevated Cdkn1b, Ddit3, Gas2l1,Trp53inp1, and Gadd45a mRNAs, validating the array results. In addition,the Exosc9 knockdown significantly upregulated the same mRNAs,suggesting that the exosome complex suppresses these RNAs encoding cellcycle regulatory proteins (FIG. 38). In primary human erythroblasts,GATA-1 occupies intronic sites at CDK1N1B, GAS2L1, and GADD45A andpromoter regions of CDK1N1B, DDIT3, GAS2L1, and GADD45A, suggestingdirect GATA-1 regulation of these genes (FIG. 39). The Exosc8- andExosc9-mediated suppression of genes mediating G₁ cell cycle arrest uponExosc8 knockdown reveals insights regarding how exosome complexcomponents control the erythroid cell cycle.

GATA-1 directly represses Exosc8 expression during maturation, thusindirectly upregulating Exosc8 targets. Given that GATA-1 also directlyactivates these Exosc8-repressed genes, this suggests that GATA-1/Foxo3abrogates the Exosc8-mediated suppression, thus activating genes thatpromote erythroid maturation, including those involved in cell cyclearrest and hemoglobin synthesis (FIG. 40).

Example 9 Role of Additional Exosome Components to Control Autophagy

If Exosc8 functions through the exosome complex to control autophagy anderythroid gene expression, reducing the levels of additional exosomecomplex components should phenocopy the Exosc8 knockdown. Exosc10, Dis3,and Dis3l were knocked down (FIG. 41) and it was tested whether theyresemble Exosc8 in regulating gene expression in G1E-ER-GATA-1 erythroidcells. The Exosc10 knockdown had a similar or quantitatively greaterinfluence on autophagy gene expression versus the Exosc8 knockdown (FIG.42). Resembling the RNA analysis, Western blotting also revealedsignificantly increased LC3-II upon Exosc8 or Exosc10 knockdown (FIG.43), indicating increased autophagy. Knocking-down Exosc10 alsoincreased βmajor and Alas2 expression without affecting Fog-1 or Gata2expression (FIG. 44). Atg16l1, which is not GATA-1-regulated, wasunaffected by the knockdown, analogous to the Exosc8 knockdown (FIG.22). Reducing Dis3 or Dis3l levels either did not significantly alterthe gene expression or slightly increased expression (FIGS. 42 and 44).The similar Exosc8 and Exosc10 activities implicate the exosome complexin selectively mediating gene expression changes and in inducingautophagy.

Discussion: Described herein is a novel genetic network regulatedcombinatorially by two proteins that control erythropoiesis, GATA-1 andFoxo3. In addition to GATA-1/Foxo3-activated autophagy genes, thisnetwork includes GATA-1/Foxo3-repression of Exosc8. Functional analysesestablished a mechanism in which GATA-1/Foxo3 pro-erythroid maturationactivity is opposed by exosome complex components. By downregulatingthese components, GATA-1/Foxo3 overcome a barrier to erythroid cellmaturation.

Knocking down Exosc8 or Exosc9 resulted in upregulation of genesencoding proteins that control multiple aspects of erythroid maturation.Morphological and flow cytometric analysis provided strong evidence thatExosc8/Exosc9 repress maturation of primary erythroid precursor cells.Exosc8/Exosc9 do not regulate GATA-1 and FOG-1 expression, excluding apotentially simple mechanism to alter GATA-1 target gene expression.Exosc8 does not exclusively function by suppressing erythroid cellmRNAs, as it also regulates primary transcripts and suppresses pSer5-PolIl occupancy at erythroid gene promoters. These results demonstrate thatExosc8 function in erythroid cells can regulate erythroid genetranscription. In Drosophila, exosome complex components colocalize withchromatin insulator factors at boundary elements and also localize topromoters. In yeast, the exosome complex localizes to nucleoli andsubnuclear regions enriched in actively transcribed genes. The exosomecomplex can regulate gene expression through alternative transcriptionstart site selection and may directly impact heterochromatin formationand gene silencing through post-transcriptional regulation.

GATA-1/Exosc8 co-regulate genes involved in cell cycle arrest,especially those that establish G₁ arrest. Cell cycle arrest in G₁ isone of the hallmarks of late-stage erythroblast differentiation.Enforced cell proliferation of late-stage bone marrow erythroblastsleads to decreased hemoglobin production, retention of mitochondria, andapoptosis. Cdkn1b (p27^(kip1)), which is GATA-1-activated andExosc8-repressed, is a highly studied cell cycle arrest protein. Cdkn1bcontrols the levels and activity of cyclin-dependent kinases (CDK2, CDK4and CDK6), which bind Cyclin D and Cyclin E, inducing G₁ arrest inmultiple cell types. Cdkn1b appears to be important for erythroidmaturation, as it is induced by GATA-1 and accumulates during terminalmaturation of erythroblasts. Furthermore, ectopic expression of Cdkn1bpromotes erythroid maturation of K562 erythroleukemia cells. Althoughthe other GATA-1-activated, Exosc8-repressed G₁ arrest genes had notbeen linked to erythropoiesis, based on their established functions inother systems, we predict that these genes contribute to G₁ arrestduring erythroid maturation.

GATA-1 directly upregulates Foxo3 expression and then functions incombination with Foxo3 to repress Exosc8 expression. Exosc8, as acomponent of the exosome complex, establishes a blockade to erythroidmaturation by suppressing GATA-1-activated genes that contribute toessential erythroid maturation processes including hemoglobin synthesis,cell cycle arrest, and other key processes. Reduced expression of theexosome complex components, either physiologically throughGATA-1/Foxo3-mediated co-repression, or experimentally, eliminates thisblockade, allowing thus facilitating erythroid maturation (FIG. 40).

Foxo3 and GATA-2 are important regulators of hematopoietic stem cellsand neural stem/progenitor cells. Considering certain similaritiesbetween GATA-1 and GATA-2, it is attractive to propose that GATA-2 mayalso function with Foxo3 to establish genetic networks in distinct celltypes. GATA factor-Foxo3 interactions may therefore have broaderfunctional roles in the context of stem cell biology.

In summary, the discoveries of novel genetic networks and unexpectedbiological functions of exosome complex components described hereinprovide new mechanistic insights into the essential process of red bloodcell development. Key nodal points in the respective mechanisms will bevulnerable to perturbations that cause and/or contribute to human redcell disorders, and this work establishes a new framework forcontrolling erythroid maturation.

As used herein, “nucleic acid” means single-, double-, ormultiple-stranded DNA, RNA and derivatives thereof. In certainembodiments, the nucleic acid is single stranded. Modifications mayinclude those that provide other chemical groups that incorporateadditional charge, polarizability, hydrogen bonding, electrostaticinteraction, and functionality to the nucleic acid. Such modificationsinclude, but are not limited to, phosphodiester group modifications(e.g., phosphorothioates, methylphosphonates), 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping moieties. A2′deoxy nucleic acid linker is a divalent nucleic acid of anyappropriate length and/or internucleotide linkage wherein thenucleotides are 2′deoxy nucleotides.

Certain nucleic acid compounds can exist in unsolvated forms as well assolvated forms, including hydrated forms. In general, the solvated formsare equivalent to unsolvated forms. Certain nucleic acid compounds mayexist in multiple crystalline or amorphous forms. In general, allphysical forms are equivalent for the uses contemplated by the methodsprovided herein.

Certain nucleic acid compounds may possess asymmetric carbon atoms(optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers.

In general, complementary nucleic acid strands hybridize under stringentconditions. The term “stringent hybridization conditions” or “stringentconditions” refers to conditions under which a nucleic acid hybridizesto an inhibitory nucleic acid, for example, to form a stable complex(e.g. a duplex), but to a minimal number of other sequences. Thestability of complex is a function of salt concentration and temperature(See, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual 2d Ed. (Cold Spring Harbor Laboratory, (1989); incorporatedherein by reference). Stringency levels used to hybridize a nucleic acidto an inhibitory nucleic acid can be readily varied by those of skill inthe art. The phrase “low stringency hybridization conditions” refers toconditions equivalent to hybridization in 10% formamide, 5 timesDenhart's solution, 6 times SSPE, 0.2% SDS at 42° C., followed bywashing in 1 times SSPE, 0.2% SDS, at 50° C. Denhart's solution and SSPEare well known to those of skill in the art as are other suitablehybridization buffers. (See, e.g., Sambrook et al.). The term“moderately stringent hybridization conditions” refers to conditionsequivalent to hybridization in 50% formamide, 5 times Denhart'ssolution, 5 times SSPE, 0.2% SDS at 42° C., followed by washing in 0.2times SSPE, 0.2% SDS, at 60° C. The term “highly stringent hybridizationconditions” refers to conditions equivalent to hybridization in 50%formamide, 5 times Denhart's solution, 5 times SSPE, 0.2% SDS at 42° C.,followed by washing in 0.2 times SSPE, 0.2% SDS, at 65° C.

“Complementary,” as used herein, refers to the capacity for precisepairing of two nucleobases (e.g. A to T (or U), and G to C) regardlessof where in the nucleic acid the two are located. For example, if anucleobase at a certain position of nucleic acid is capable of hydrogenbonding with a nucleobases at a certain position of an inhibitorynucleic acid, then the position of hydrogen bonding between the nucleicacid and the inhibitory nucleic acid is considered to be a complementaryposition. The nucleic acid and inhibitory nucleic acid are“substantially complementary” to each other when a sufficient number ofcomplementary positions in each molecule are occupied by nucleobasesthat can hydrogen bond with each other. Thus, the term “substantiallycomplementary” is used to indicate a sufficient degree of precisepairing over a sufficient number of nucleobases such that stable andspecific binding occurs between the nucleic acid and an inhibitorynucleic acid. The phrase “substantially complementary” thus means thatthere may be one or more mismatches between the nucleic acid and theinhibitory nucleic acid when they are aligned, provided that stable andspecific binding occurs. The term “mismatch” refers to a site at which anucleobases in the nucleic acid and a nucleobases in the inhibitorynucleic acid with which it is aligned are not complementary. The nucleicacid and inhibitory nucleic acid are “perfectly complementary” to eachother when the nucleic acid is fully complementary to the inhibitorynucleic acid across the entire length of the nucleic acid.

Generally, a nucleic acid is “antisense” to an inhibitory nucleic acidwhen, written in the 5′ to 3′ direction, it comprises the reversecomplement of the corresponding region of the target nucleic acid.“Antisense compounds” are also often defined in the art to comprise thefurther limitation of, once hybridized to a target, being able tomodulate levels, expression or function of the target compound.

As used herein, “sequence identity” or “identity” refers to thenucleobases in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. As used herein,“percentage of sequence identity” means the value determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When nucleic acid compounds containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When nucleic acid compounds contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Certain nucleic acid compounds containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the nucleic acid compounds may be regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the compound together with a pharmaceuticallyacceptable excipient, such as diluents, preservatives, solubilizers,emulsifiers, and adjuvants. As used herein “pharmaceutically acceptableexcipients” are well known to those skilled in the art.

Tablets and capsules for oral administration may be in unit dose form,and may contain conventional excipients such as binding agents, forexample syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tabletting lubricant, forexample magnesium stearate, talc, polyethylene glycol or silica;disintegrants for example potato starch, or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in normal pharmaceutical practice. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavoring or coloring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art.Topical administration includes transdermal formulations such aspatches.

For topical application to the eye, the inhibitor may be made up into asolution or suspension in a suitable sterile aqueous or non-aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzalkonium chlorideor chlorhexidine, and thickening agents such as hypromellose may also beincluded.

The active ingredient may also be administered parenterally in a sterilemedium, either subcutaneously, or intravenously, or intramuscularly, orintrasternally, or by infusion techniques, in the form of sterileinjectable aqueous or oleaginous suspensions. Depending on the vehicleand concentration used, the drug can either be suspended or dissolved inthe vehicle. Advantageously, adjuvants such as a local anaesthetic,preservative and buffering agents can be dissolved in the vehicle.

Pharmaceutical compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. The term “unit dosage” or “unit dose” means a predeterminedamount of the active ingredient sufficient to be effective for treatingan indicated activity or condition. Making each type of pharmaceuticalcomposition includes the step of bringing the active compound intoassociation with a carrier and one or more optional accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidor solid carrier and then, if necessary, shaping the product into thedesired unit dosage form.

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second etc.as used herein are not meant to denote any particular ordering, butsimply for convenience to denote a plurality of, for example, layers.The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

1. A method of enhancing erythropoiesis in an individual in needthereof, comprising administering an effective amount of an agent thatdecreases the expression of Exosc9, wherein the agent produces anincrease in red blood cell production in the individual, and wherein theagent is an inhibitory nucleic acid molecule that selectively decreasesthe expression of Exosc9.
 2. The method of claim 1, wherein theindividual is a human in need of treatment for an anemic disorder,hemophilia, thalassemia, sickle cell disease, bone marrowtransplantation, hematopoietic stem cell transplantation,thrombocytopenia, pancytopenia, or hypoxia.
 3. The method of claim 2,wherein the anemic disorder is associated with aging, infectiousdisease, chronic renal failure, end-stage renal disease, renaltransplantation, cancer, AIDS, antiviral therapy, chronic stress,chemotherapy, radiation therapy, bone marrow transplantation,nutritional iron deficiency, blood-loss, hemolysis, or is of geneticorigin.
 4. The method of claim 3, wherein the individual has a reducedhematocrit.
 5. The method of claim 1, wherein the inhibitory nucleicacid molecule is a small interfering RNA, an antisense RNA, a ribozyme,or a triple helix nucleic acid.
 6. The method of claim 5, wherein thesmall interfering RNA is an siRNA or an shRNA.
 7. The method of claim 6,wherein the small interfering RNA comprises 19 to 29 nucleotides thatare substantially complementary to a sequence of 19 to 29 nucleotides ofExosc9.


8. The method of claim 7, wherein Exosc9 has SEQ ID No. 2, 3 or
 10. 9.The method of claim 7, wherein the small interfering RNA is an shRNAhaving SEQ ID NO. 23 or 24, the corresponding sequence of SEQ ID NO. 2,3, or a combination thereof.